| 溶藻弧菌抗独特型抗体单链抗体在毕赤酵母中的表达 |
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| 引用本文： | 付建芳,涂艳阳,秦红,黄威权. 溶藻弧菌抗独特型抗体单链抗体在毕赤酵母中的表达[J]. 免疫学杂志, 2009, 0(5) |
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| 作者姓名： | 付建芳 涂艳阳 秦红 黄威权 |
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| 作者单位： | 第四军医大学西京医院内分泌科;解放军63870部队医院;第四军医大学组织胚胎学教研室; |
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| 基金项目： | 国家高技术研究发展计划(863)资助项目(2006AA100308) |
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| 摘 要： | 目的构建溶藻弧菌抗独特型抗体的单链抗体(scFv)基因的毕赤酵母分泌型表达载体,并对其表达的蛋白进行免疫原性鉴定。方法从分泌溶藻弧菌抗独特型抗体的杂交瘤细胞株(2F4)中克隆该抗体的重链可变区(VH)基因和轻链可变区(VL)基因(GenBank accession No:DQ011530和DQ011531),并通过基因重组为scFv基因。然后将其克隆入毕赤酵母菌分泌型表达载体pPIC9K中,经序列测定后,电转化入毕赤酵母菌SMD1168中,将经表型筛选和G418抗性筛选的重组酵母菌株进行PCR鉴定。以甲醇进行诱导表达,表达后的重组蛋白利用动物免疫试验进行免疫原性鉴定。结果获得毕赤酵母菌分泌型表达载体pPIC9K-scFv,经G418浓度梯度筛选及PCR鉴定得到高拷贝整合的重组酵母菌株,ELISA测定和动物免疫试验显示抗独特性抗体的scFv具有与溶藻弧菌一样的免疫原性。结论构建溶藻弧菌抗独特型抗体的scFv基因的真核表达载体并成功表达,为进一步研究其生物学活性奠定了基础。
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| 关 键 词： | 溶藻弧菌 单链抗体 真核表达 毕赤酵母菌 |
Immunogenicity of single chain variable fragment of anti-idiotype monoclonal antibody against Vibrio alginolyticus and its expression in Pichia pastoris |
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| FU Jianfang,TU Yanyang,QIN Hong,HUANG Weiquan. Immunogenicity of single chain variable fragment of anti-idiotype monoclonal antibody against Vibrio alginolyticus and its expression in Pichia pastoris[J]. Immunological Journal, 2009, 0(5) |
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| Authors: | FU Jianfang TU Yanyang QIN Hong HUANG Weiquan |
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| Affiliation: | FU Jianfang,TU Yanyang,QIN Hong,HUANG Weiquan Department of Endocrinology,Xijing Hospital,Fourth Military Medical University,Xi'an 710032,China |
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| Abstract: | Objective To construct recombinant Pichia pastoris secretion type expression vector and characterize immunogenicity of single chain variable fragment(scFv)of anti-idiotype monoclonal antibody against Vibrio alginolyticus.Methods Total RNA was extracted from hybridoma cell 2F4 secreting mAb against Vibrio alginolyticus,and then cDNA was amplified by RT-PCR.Then the RT-PCR product was recombined and inserted into secretion type expression vector pPIC9K-scFv.Recombinant vector was transformed into Pichia pasto... |
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| Keywords: | Vibrio alginolyticus single-chain antibody eukaryotic expression Pichia pastoris |
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